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UNIVERSITI PUTRA MALAYSIA

ISOLATION, OPTIMIZATION AND RECOVERY OF ASTAXANTHIN FROM SHRIMP SHELL WASTES OF LOCALLY ISOLATED AEROMONAS

HYDROPHILA (STEINER)

CHEONG JEE YIN

FS 2016 60

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ISOLATION, OPTIMIZATION AND RECOVERY OF ASTAXANTHIN FROM

SHRIMP SHELL WASTES OF LOCALLY ISOLATED Aeromonas hydrophila

(Steiner)

By

CHEONG JEE YIN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Doctor of Philosophy

September 2016

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for non-commercial purposes from the copyright holder. Commercial use of any

material may only be made with the express, prior, written permission of Universiti

Putra Malaysia.

Copyright © Universiti Putra Malaysia

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Doctor of Philosophy

ISOLATION, OPTIMIZATION AND RECOVERY OF ASTAXANTHIN FROM

SHRIMP SHELL WASTES OF LOCALLY-ISOLATED Aeromonas hydrophila

(Steiner)

By

CHEONG JEE YIN

September 2016

Chairman : Muskhazli Mustafa, PhD

Faculty : Science

Astaxanthin is a red pigment naturally produced by microalgae. Being part of the

shrimp diet, this pigment was accumulated in their body, shells and eggs. The total

amount of shell wastes discarded during processing could reach up to 40-50% of its

body weight. Previously, astaxanthin was recovered from shrimp shells using lactic

acid bacteria fermentation. This method promises a good return but has higher

maintenance. Meanwhile aerobic bacteria fermentation has less maintenance and was

less explored in this context. Astaxanthin in its free form is readily oxidised while

astaxanthin in crustaceans appears to be in complexes (carotenoprotein,

carotenolipoprotein and chitinocarotenoids) which is less prone to oxidation. The

current aim was to recover astaxanthin from shrimp shell wastes through aerobic

bacteria fermentation. In order to obtain astaxanthin from shrimp wastes, chitinase and

protease were crucial to dechitinize and deproteinize the pigment from its stable

complex structures. In this study, the first objective was to isolate potential shrimp shell

degrading bacteria from shrimp shells. Bacteria were isolated from shrimp shells and

screened using shrimp crab shell powder. A total of 19 isolates producing chitinase and

protease were obtained. Potential isolates were then compared among each other using

shrimp shell waste powder (SSWP) to seek for an optimum enzyme (chitianse and

protease) producing isolate. The selected isolate was identified as Aeromonas

hydrophila. Naturally, shrimp shells are calcified and may hinder further recovery of

astaxanthin. Hence, the addition of cell disruptions was aimed to loosen the complex

structure of shrimp shells. A dual effect “shell disruption” method was adopted where

non-chemical shell disruption was applied on the wastes as pre-treatment followed by

microbial enzyme disruption as the second. The amount of astaxanthin recorded after

the application of shell disruptions was comparable between treatments of control (non-

pretreated SSWP fermented with microbial enzyme) and autolysis (pre-treated SSWP

with autolysis and microbial enzyme). The control treatment has resulted in 2.3 ±

0.1179µg/ml of astaxanthin recovery, while combined autolysis treatment and

microbial enzyme yielded 2.11 ± 0.0961µg/ml. Since both treatments gave similar yield

and were not significantly different, single shell disruption (control treatment) was

sufficient to produce a good recovery. In order to enhance enzyme production and

astaxanthin recovery, the culture media and conditions were also optimized.

Optimization of the culture media and conditions was carried out using Response

Surface Methodology analysis (RSM). The media was screened with various media

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supplements (nitrogen, inorganic salts and carbon sources) and concentrations (1, 3, 5,

7, 9% w/v) before optimizing with RSM. An optimum media containing 3% MSG, 1%

glucose, pH 7.0 and 30 °C with a constant supply of 0.1% K2HPO4, 0.05%

MgSO4.7H2O, and 9% SSWP was used to culture A. hydrophila. In comparison, by

using the optimum culture astaxanthin recovery, chitinase and protease activity has

increase up to 38.4%, 30% and 36% respectively as compared to un-optimized media.

To achieve the main goal of this investigation, carotenoids were purified with thin layer

chromatography (TLC) and the presence of astaxanthin was confirmed using high

pressure liquid chromatography (HPLC). Carotenoids were obtained after bacteria

fermentation on SSWP under optimized conditions and were soxhlet extracted with

acetone and concentrated before subjecting to TLC. The best mobile phase in

separating the pigments was hexane: acetone (3:1 v/v). The potential band for

astaxanthin was obtained from TLC at Rf value of 0.33. This band was re-extracted in

acetone and subjected to confirmation using HPLC with a reference standard. The

presence of astaxanthin was confirmed at retention time of 15.5, 16.4, 17.4, and 18.3

minutes. In conclusion, astaxanthin can be recovered from shrimp shell waste with

aerobic fermentation of A. hydrophila.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PENGASINGAN, PENGOPTIMUMAN DAN PEMEROLEHAN SEMULA

ASTAXANTHIN DARIPADA SISA KULIT UDANG OLEH ISOLAT-

TEMPATAN Aeromonas hydrophila (Steiner)

Oleh

CHEONG JEE YIN

September 2016

Pengerusi : Muskhazli Mustafa, PhD

Fakulti : Sains

Astaxanthin adalah pigmen merah semulajadi yang dihasilkan oleh mikroalga. Sebagai

sebahagian daripada diet udang, pigmen ini sering terkumpul di dalam badan, kulit dan

telur. Jumlah kulit udang yang dibuang semasa pemprosesan boleh mencecah sehingga

40-50% daripada beratnya. Sebelum ini, pemulihan semula astaxanthin daripada kulit

udang adalah secara penapaian dengan bakteria asid laktik. Kaedah ini menjanjikan

hasil yang lumayan, tetapi mempunyai kos penyelenggaraannya tinggi. Sementara itu,

penapaian bakteria secara aerobik yang kurang penyelenggaraan kurang diterokai.

Astaxanthin dalam struktur bebas adalah lebih cenderung untuk dioksidakan manakala

astaxanthin yang wujud dalam krustacea merupakan astaxanthin dalam struktur

kompleks (karotenoprotein, karotenolipoprotein dan khitinokarotenoid) yang kurang

cenderung pada pengoksidaan. Tujuan utama penyelidikan ini adalah untuk

menulenkan astaxanthin daripada sisa kulit udang melalui cara penapaian bakteria

aerobic. Dalam usaha untuk mendapatkan astaxanthin daripada sisa udang, kitinase dan

protease adalah penting proses pembebasan khitin dan protein daripada struktur pigmen

yang stabil dan kompleks. Dalam kajian ini, objektif pertama adalah untuk

mengasingkan bakteria yang berpotensi untuk degradasi sisa kulit udang daripada kulit

udang. Bakteria yang berjaya diasingkan daripada kulit udang disaring menggunakan

serbuk kulit ketam. Sebanyak 19 isolat yang menghasilkan kitinase dan protease telah

diperolehi. Isolat yang berpotensi dibandingkan antara satu sama lain dengan

menggunakan serbuk sisa kulit udang (SSWP) bagi mendapatkan isolat yang paling

optimum dalam penghasilan enzim (kitinase dan protease). Isolat yang terpilih telah

dikenal pasti sebagai Aeromonas hydrophila. Secara semulajadi, kulit udang

mengalami proses kalsifikasi yang menyukarkan bagi mendapatkan lebih astaxanthin.

Oleh itu, penambahan kaedah gangguan sel bertujuan untuk melonggarkan struktur

kompleks kulit udang. Kaedah Dwi kesan ‘ganguan kulit’ digunakan dimana

‘gangguan kulit’ tanpa bahan kimia dikenakan pada sisa-sisa sebagai pra-rawatan dan

diikuti oleh gangguan enzim mikrob dijadikan fasa kedua. Jumlah astaxanthin

direkodkan selepas gangguan kulit adalah setanding antara rawatan kawalan (tiada pra-

rawatan penapaian SSWP dengan enzim mikrob) dan autolisis (pra-rawatan SSWP

dengan autolisis dan enzim mikrob). Rawatan kawalan telah memberikan perolehan

astaxanthin sebanyak 2.3 ± 0.1179μg/ml manakala gabungan rawatan autolisis dan

enzim mikrob menghasilkan 2.11 ± 0.0961μg/ml. Oleh kerana, kedua-dua rawatan

memberikan hasil statiistik yang tidak signifikasi, gangguan secara tunggal (rawatan

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kawalan) adalah memadai untuk menghasilkan perolehan yang baik. Dalam usaha

untuk meningkatkan pengeluaran enzim dan pemulihan astaxanthin , media kultur dan

keadaan telah dioptimumkan. Pengoptimuman kultur media dan penyediaan bakteria

telah dilaksanakan dengan menggunakan analisis kaedah response surface (RSM).

Kultur media telah disaring dengan pelbagai jenis bahan tambahan (sumber nitrogen,

garam inorganic dan karbon) dan kepekatan (1, 3, 5, 7, 9% w/v) sebelum dioptimisasi

dengan RSM. Media optima yang mengandungi 3% MSG, 1% glukosa, pH 7,0 dan 30

° C dengan bekalan malar 0.1% K2HPO4 , 0.05% MgSO4.7H2O , dan 9% SSWP untuk

kultur A. hydrophila. Sebagai perbandingan, penggunaan kultur media optima telah

masing-masing memberikan pemulihan astaxanthin, peningkatan aktiviti enzim kitinase

dan protease sebanyak 38.4%, 30% dan 36% berbanding media tidak optima. Bagi

mencapai objektif utama dalam kajian ini, karotenoid telah ditulenkan dengan

menggunakan kaedah kromatografi lapis tipis (TLC) dan kehadiran astaxanthin

disahkan dengan menggunakan kromatografi cecair tekanan tinggi (HPLC). Karotenoid

yang diperolehi selepas penapaian dengan bakteria dalam media optimum diekstrak

dengan aseton menggunakan kaedah soxhlet dan dipekatkan sebelum digunakan dalam

TLC. Campuran heksana : aseton (3: 1 v / v) adalah sistem eluen terbaik untuk

pemisahan pigmen. Kehadiran astaxanthin dapat telah ditunjukkan oleh jalur yang

terbentuk dari TLC pada nilai Rf 0.33 . Jalur ini kemudiannya diekstrak semula dengan

aseton dan digunakan untuk pengesahan astaxanthin menggunakan HPLC dengan

perbandingan kepada bahan rujukan piawaian. Astaxanthin disahkan kehadirannya

pada sela masa minit ke 15.5, 16.4, 17.4 dan 18.3. Kesimpulannya, astaxanthin dapat

diperolehi semula daripada sisa kulit udang melalui penapaian aerobik dengan A.

hydrophila.

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ACKNOWLEDGEMENTS

My greatest gratitude goes out to my supervisor Assoc. Prof. Dr. Muskhazli Mustafa,

for his never ending support, trust, guidance and understanding in completing this

project. To both of my co-supervisors Assoc. Prof. Dr. Nor Azwady Abd Aziz and Dr.

Siti Aqlima Ahmad, thank you for the support and guidance provided throughout the

project. This thesis would not been produced without your wise guidance, knowledge

and support.

For their generous assistance in this research, I would like to express my gratitude to

my lab mates and seniors, Noormasshela, Nurul Shaziera, Nithyaa Perumal, Safiya

Yakubu, Salihu, Teng Suk Kuan and Chew Weiyun. A special thanks to my lab

assistants, Mrs. Azleen and Mrs. Farah for their assistance in chemicals and

instruments for use.

I would also like to extend my gratitude to those who have supported me during my

conversion and all the way towards the completion of this thesis especially Dr.

Christina Yong, Dr. Amal, Dr. Hishamuddin Omar, Dr. Syaizwan, Prof. Dr. Rusea, Dr.

Alvin Hee, Dr. Abdul Rahim and all the lecturers in the Department of Biology. To Dr.

Issam, Dr. Shuukri and Dr.Yunus; thank you for the extra knowledge in spectroscopy

and protein purification.

To my family, thank you for your love, patience and believe. To my friends, thank you

for the joyful and happy times together and encouragements within the past 4 years.

Finally I would like to express my gratefulness to the Laboratory of Plant Systematic

and Microbe, Department of Biology and Faculty of Science, UPM for a wonderful

time and an opportunity to gain knowledge. A special thanks to the head of department,

all the staff in this department, friends and to my laboratory suppliers for providing me

all the needs.

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I certify that a Thesis Examination Committee has met on 6 September 2016 to conduct

the final examination of Cheong Jee Yin on her thesis entitled “Isolation, Optimization

and Recovery of Astaxanthin from Shrimp Shell Wastes of Locally-Isolated

Aeromonas hydrophila (Steiner)” in accordance with the Universities and University

Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106]

15 March 1998. The Committee recommends that the student be awarded the Doctor of

Philosophy.

Members of the Thesis Examination Committee were as follows:

Rusea Go, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Hishamuddin bin Omar, PhD

Senior Lecturer

Faculty of Science


(Internal Examiner)

Shuhaimi bin Mustafa, PhD

Professor

Halal Products Research Institute


(Internal Examiner)

Jasim Ahmed, PhD

Senior Lecturer

Kuwait Institute for Scientific Research

Kuwait

(External Examiner)

NOR AINI AB. SHUKOR, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 3 November 2016

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The

members of the Supervisory Committee were as follows:

Muskhazli Mustafa, PhD

Associate Professor

Faculty of Science


(Chairman)

Nor Azwady Abd Aziz, PhD

Associate Professor

Faculty of Science


(Member)

Siti Aqlima Ahmad, PhD

Senior Lecturer

Faculty of Biotechnology and Biomolecular Sciences


(Member)

BUJANG KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other degree

at any other institutions;

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and Innovation) before thesis is published (in the form

of written, printed or in electronic form) including books, journals, modules,

proceedings, popular writings, seminar papers, manuscripts, posters, reports,

lecture notes, learning modules or any other materials as stated in the Universiti

Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: Date:

Name and Matric No.: Cheong Jee Yin (GS 30313)

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature:

Name of

Chairman of

Supervisory

Committee:

Assoc. Prof. Dr. Muskhazli Mustafa

Signature:

Name of Member

of Supervisory

Committee:

Assoc. Prof. Dr. Nor Azwady Abd Aziz

Signature:

Name of Member

of Supervisory

Committee:

Dr. Siti Aqlima Ahmad

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION vii

LIST OF TABLES xiii

LIST OF FIGURES xiv

LIST OF APPENDICES xvi

LIST OF ABBREVIATIONS xvii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Astaxanthin 5

2.1.1 Structures of astaxanthin 5

2.1.2 Abundance of astaxanthin 6

2.1.3 Uses of Astaxanthin 8

2.1.4 Extraction of astaxanthin 9

2.2 Microbial enzyme hydrolysis of crustacean wastes 10

2.3 The genus Aeromonas 11

2.3.1 Aeromonas hydrophila 12

2.3.2 Secretions of the genus Aeromonas 13

2.3.2.1 Amylase 13

2.3.2.2 Lipase 14

2.3.2.3 β-lactamase 14

2.3.2.4 Protease 15

2.3.2.5 Chitinase 15

2.3.3 Pathogenicity and virulence of Aeromonads 15

2.3.4 Resistance of Aeromonas towards antibiotics 16

2.4 Crustacean wastes and its valuable substances 17

2.4.1 Chitin and chitosan 18

2.4.2 Economy of valuables 19

2.4.3 Managing with chemical treatments 20

2.5 Cell Disruptions 21

2.5.1 Chemical cell disruptions 21

2.5.2 Physical cell disruptions 21

2.5.2.1 Mechanical cell disruptions 21

2.5.2.2 Non mechanical cell disruptions 21

2.5.3 Enzymatic cell disruptions 22

2.6 Response surface methodology 22

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3 AEROMONAS HYDROPHILA AS A SUITABLE

CHITINASE AND PROTEASE PRODUCING BACTERIA

ISOLATED FROM SHRIMP SHELL WASTES

3.1 Introduction 24

3.2 Materials and Methods 25

3.2.1 Preparation of shrimp shell wastes 26

3.2.2 Preparation of colloidal chitin 27

3.2.3 Isolation of bacteria 27

3.2.4 Comparison of possible bacteria isolates 27

3.2.5 Enzyme assays 28

3.2.6 Morphology of the isolated bacteria 28

3.2.7 Molecular 16s RNA sequencing 29

3.2.8 Data analysis 29

3.3 Results and Discussions 29

3.3.1 Isolations of bacteria and enzyme evaluations 30

3.3.2 Comparison of selected isolates in shrimp shell

wastes utilization

31

3.3.3 Identification of the selected bacteria 34

3.4 Conclusion 38

4 THE APPLICATION OF DUAL EFFECT “SHELL

DISRUPTIONS” ON ENHANCING ASTAXANTHIN

RECOVERY FROM SHRIMP SHELL WASTE

4.1 Introduction 39


4.2.1 Preparation of shrimp shells 42

4.2.2 Dual effect shell disruption 42

4.2.2.1 Physical shell disruptions as pre-

treatment to shrimp shell wastes

42

4.2.2.2 Microbial enzymatic shell disruption 42

4.2.3 Analysis of astaxanthin 43

4.2.4 Enzyme analysis 43


4.3.1 Effects of cell disruptions on astaxanthin

availability

44

4.3.2 Availability of astaxanthin through enzymatic cell

disruption

46

4.4 Conclusion 50

5 ENHANCEMENT OF AEROMONAS HYDROPHILA

ENZYME PRODUCTION, GROWTH AND

ASTAXANTHIN RECOVERY BY OPTIMIZATION OF

THE CULTURE MEDIA

5.1 Introduction 51


5.2.1 Screening of sources with SFO technique 54

5.2.2 Screening for temperature and pH 55

5.2.3 Colony Forming Units 55

5.2.4 Experimental Design 56


5.3.1 Selection of physio-chemical conditions 57

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5.3.1.1 Nitrogen sources 57

5.3.1.2 Inorganic salts 61

5.3.1.3 Carbon sources 63

5.3.1.4 Temperature 66

5.3.1.5 pH 68

5.3.3 Optimization of the culture media and conditions 69

5.3.3.1 Enzyme activity 71

5.3.3.2 Astaxanthin yield 78

5.3.4 Validation of the model 82

5.4 Conclusion 84

6 CONFIRMATION OF ASTAXANTHIN EXTRACTED

FROM SHRIMP SHELL WASTES AFTER MICROBIAL

FERMENTATION

6.1 Introduction 85


6.2.1 Preparation of carotenoids 88

6.2.2 Extraction of carotenoids 88

6.2.3 Purification of carotenoids by thin layer

chromatography (TLC)

88

6.2.4 HPLC confirmation of the presence of astaxanthin 89


6.3.1 Separation of carotenoids extract 90

6.3.2 Confirmation of astaxanthin presence 94

6.4 Conclusion 99

7 GENERAL DISCUSSION AND FUTURE

RECOMENDATIONS

100

REFERENCES 108

APPENDICES 145

BIODATA OF STUDENT 163

LISTS OF PUBLICATIONS 164

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LISTS OF TABLES

Table Page

3.1 Top 10 BLAST results of gene sequenced by S 010 taken from the

gene bank

35

4.1 Availability of astaxanthin from SSW when treated with

mechanical cell disruptions

44

4.2 Astaxanthin availability after fermentation with A. hydrophila on

PTSW

47

5.1 Independent variables and their coded values 56

5.2 Astaxanthin recovery after incubation and growth of A. hydrophila

in various temperatures

67

5.3 Enzyme activities, astaxanthin yield and growth of A. hydrophila

in different pHs

68

5.4 Experimental design and the results of CCD for the enzyme

production and astaxanthin yield from the fermentation with A.

hydrophila

70

5.5 Regression analysis of protease and chitinase from the CCD

response surface model

71

5.6 Analysis of variance (ANOVA) for protease activity 72

5.7 Analysis of variance (ANOVA) for chitinase activity 75

5.8 Regression analysis of astaxanthin yield from the CCD response

surface model

78

5.9 Analysis of variance (ANOVA) for astaxanthin yield 79

5.10 Predicted responses value by CCD and actual experimental value

under the suggested optimum conditions

82

6.1 Internationally accepted Rf values of astaxanthin 93

6.2 Retention time for similar peaks in band 2 and standard 97

7.1 Comparison of protease and chitinase production of various

bacteria

103

7.2 Comparison of different extraction methods in obtaining

astaxanthin from various sources

104

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LISTS OF FIGURES

Figure Page

2.1 Chemical structure of 3,3’-dihydroxy-ß-carotene-4,4’-dione

(astaxanthin)

5

2.2 Chemical structure of chitin (2-acetamido-2-deoxy-D-glucose). 18

2.3 Chemical structure of chitosan 19

3.1 Flow chart of the experimental design 26

3.2 Chitinase and protease activity of isolated samples 31

3.3 Comparison of isolate S 010 and L 001 for their enzymatic

activity cultured in minimal synthetic media supplied with

SSWP

32

3.4 Negatively stained and coccobacilli shaped isolate S 010 viewed

under 400X magnification (a) and 1000X magnification (b)

36

4.1 Schematic diagram on various mechanical shell disruptions and

bacterial fermentation

41

4.2 Chitinase and protease activity of PTSW fermented with A.

hydrophila

48

5.1 Schematic diagram on the selection of sources and condition for

optimization of A. hydrophila with response surface

methodology

54

5.2 Enzyme activities of A. hydrophila cultured in different nitrogen

sources and concentration. (B.P- bacto peptone; MSG-

monosodium glutamate; Y.E.- yeast extract; S.N- sodium

nitrate)

57

5.3 Astaxanthin yield from shrimp shells cultured in different

nitrogen sources and concentrations

58

5.4 Enzyme activities, astaxanthin availability and growth of A.

hydrophila in different concentrations of MSG

60

5.5 Enzyme activities of A. hydrophila cultured in different

inorganic salt and concentrations. (NaCl- Sodium chloride;

CaCl- Calcium chloride; CuSO- Copper (II) sulphate).

62

5.6 Astaxanthin availability in various inorganic salts in different

concentrations

62

5.7 Enzyme activities of A. hydrophila cultured in different carbon

sources and concentrations

64

5.8 Astaxanthin availability in various carbon sources in different

concentrations

64

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5.9 Enzyme activities, astaxanthin availability and growth of A.

hydrophila in different concentrations of glucose

65

5.10 Enzyme activities of A. hydrophila at various temperatures

tested

67

5.11 Response surface 3D plots showing interactions between MSG

and pH (a), glucose and pH (b), temperature and pH (c), glucose

and MSG (d), temperature and MSG (e) and temperature and

glucose (f) on protease activity

74




glucose (f) on chitinase activity.

77




glucose (f) on astaxanthin yield

81

5.14 Comparison of protease and chitinase activity (a) and

astaxanthin yield (b) between optimized media and un-

optimized media

83

6.1 Schematic diagram for purifying carotenoids from shrimp shells

for the detection of astaxanthin presence

87

6.2 Harvested fermentation cultures that have been centrifuged (a)

and three layers were present (b). The middle layer is known as

carotenoid layer where astaxanthin was found present.

88

6.3 Thin Layer Chromatography of carotenoids developed under

different mobile phases (a) benzene: ethyl acetate (1:1), (b)

petroleum ether: acetone: diethyl amine (10: 4: 1), (c) hexane:

isopropanol (1:1) and (d) hexane : acetone (3:1)

91

6.4 Thin layer chromatography of carotenoids developed with

hexane:acetone (3:1) mobile phase in a larger amount for

confirmation of HPLC. Photographed under low light condition

92

6.5 HPLC chromatogram of astaxanthin standard (Santa Cruz

Biotechnologies). The few peaks denote different isomer present

in the standard purchased

95

6.6 Peaks of similar retention time in Band 2 (upper) and standard

(lower)

96

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LIST OF APPENDICES

Appendix Page

A Gene sequence for S 010 145

B Analysis of astaxanthin available in the water after in cell

disruption

146

C ANOVA analysis for pH responses in various buffer systems 147

D HPLC chromatogram of scrapped out bands from TLC 161

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LISTS OF ABBREVIATIONS

% percentage

°C Degrees Celsius

µg Microgram

µl Microlitre

µm Micrometre

16S/18S Svedberg units

AMW Apparent Molecular Weight

ANOVA Analysis Of Variance

APCI Atomic Pressure Chemical Ionization

ATCC American Type Cell Culture

BLAST Basic Local Alignment Search Tool

BLIS Bacteriocin-Like Inhibitory Substances

CCD Central Composite Design

CFU Colony Forming Units

cm centimetre

DAD Diode Array Detector

DMAB Dimethylamino benzoic acid

DMSO Dimethyl Sulfoxide

DNA Deoxyriboneuclotide Acid

EDTA Ethylenediaminetetraacetic Acid

ESI Electronic Spray Ionization

FAO Food And Agriculture Organization Of The United

Nations

g Gram

g Gravity

GlcNAc N-acetylglucosamine

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h Hours

HCL Hydrochloric acid

HPLC High Performance Liquid Chromatography

HSD Honest Significant Difference

K2HPO4 Dipotassium Hydrogen Phosphate

kDa Kilo Dalton

L Litre

LCMS Liquid Chromatography Mass Spectrum

m Molarity

MALDI-TOF Matrix Assisted Laser Desorption/Ionization Time Of

Flight

mg Milligram

MgSO4.7H2O Magnesium Sulphate 7-Hydrate

mm Millimetre

MSA Minimal Synthetic Agar

MSG Monosodium Glutamate

MSM Minimal Synthetic Media

NaCl Sodium Chloride

NAFLD Non-Alcoholic Fatty Liver Disease

NCBI National Center for Biotechnology Information

nm Nanometre

NMR Nuclear Magnetic Resonance

OCP Orange Carotenoid Binding Proteins

OVAT One Variable At Time

PMSF Phenylmethylsulfonyl Fluoride

PTFE Polytetraflouroethylene

CC Column Chromatography

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Rf Retention Factor

RNA Ribonucleic Acid

RPM Revolutions Per Minute

RSM Response Surface Methodology

SCSP Shrimp Crab Shell Powder

SFE Supercritical Fluid Extraction

SFO Single Factor Optimization

SPSS Statistical Package For Social Science

SSW Shrimp Shell Waste

SSWP Shrimp Shell Waste Powder

TLC Thin Layer Chromatography

USA United States of America

USD United States Dollar

UV Ultraviolet

UV-VIS Ultraviolet Visible

V Volume

w Weight

α Alpha

β Beta

λ Lambda

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CHAPTER 1

INTRODUCTION

In the recent years, the demand for food has been increasing drastically. As an

example, the aquaculture industries have incredibly grown in the past 50 years (Bostok

et al., 2010). The global production of fisheries, crustacean and mollusks according to

Fisheries Agricultural Organization has reached 91 million tons as of the year of 2012

with China being the largest producer (FAO, 2014). In food processing industries and

human consumptions, an average of 48-56% of crustacean weight is being discarded,

which includes antennae, carapaces, head, legs, shells, and tails (Pu et al., 2010). A

number of valuable recoverable substances such as chitins, carotenoids (astaxanthin)

and proteins can be recovered from these wastes. Numerous studies have shown the

presence of carotenoid with astaxanthin being the main carotenoid pigment in shrimps

(vary with species) (Sachindra et al., 2007; Shahidi et al., 1998). Being that crustacean

wastes that are left to degrade naturally or being used as land filling (Prameela et al.,

2012) are wasteful and causes environmental pollution; recycling the waste aids in

environmental issues and benefits the recovery of various valuable substances

especially astaxanthin.

Astaxanthin, (3’3-dihydroxy-β-carotene-4’4-dione) is naturally produced in microalgae

such as Haematococcus pluvialis, certain yeasts; Xanthophyllomyces dendrorhous,

(Dore and Cysewski, 2008) and the flower of the plant Adonis aestivalis (Cunningham

and Gantt, 2011) which was native to Europe. Secondarily, astaxanthin is found in

birds, fishes such as salmon, crustaceans such as krills, shrimps/prawn; Penaeus

monodon, (giant tiger prawn) crayfishes and filter feeders up taken in their dietary

supplements (Handayani et al., 2008). In certain shrimps, ingested beta-carotene can be

converted into astaxanthin by the cell’s activity (Latscha, 1990). Astaxanthin, which

provides coloration in animals varies according to the environment and its complex

structure, where it could be green, purple or blue (Sila et al., 2012) and red in cooked

crustaceans or when exposed to heat (North, 2002). The complexity of astaxanthin can

occur in three different forms in animals which are free molecules, forming complex

with protein (carotenoprotein) or lipid (carotenolipoproteins), or sterified (esters)

(Higuera-Ciapara et al., 2006).

This red pigment became important due to its unique DNA protective properties which

can be used as a supplement to boost human health (Yoshida et al., 2010). Besides

having high antioxidant properties (Capelli and Cysewski, 2007), astaxanthin is used in

various industries such as cosmetics (Seki et al., 2001), feed additives to provide

coloration (Paibulkichakul et al., 2008), and medical researches (Bhuvaneswari et al.,

2010). The importance of astaxanthin in today’s world has brought us to search for this

pigment intensity to meet the demand of various industries from the basics of

enhancing algae pigment production till genetically modified microbes to produce

natural astaxanthin (Cheng and Tao, 2012). This is because synthetically produced

astaxanthin do not give the same effect with natural astaxanthin although it fetches a

lower price of 2000 USD/kg (Ni et al., 2008) while naturally produced astaxanthin

from algae biomass fetches a higher price at 7000 USD/kg (Li et al., 2011a).

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The high price of natural astaxanthin renders a need to search for alternative ways to

feed these needing industries. An alternative way is to recycle crustacean wastes as it

contains natural astaxanthin. Previously recycling crustacean wastes was conducted

using strong acids and alkali under high temperature to increase the degradation rate

(Roberts et al., 2008). This traditional method is indeed efficient in recovering

substance such as chitin but making it almost impossible to recover other substrates

such as proteins, lipids and carotenoids (Healy et al., 2003). Furthermore, the current

method is expensive, non-environmental friendly (Rao et al., 2002) and requires extra

desalting before disposal due to high concentration of salts formed during

neutralization (Wang et al., 2006a). With the recent research advancement, managing

crustacean waste by chemicals was less preferred due to the complications arise and not

eco-friendly. Enzymatic degradation was more favorable compared to chemically treat

as the former was more environmentally friendly (Paul et al., 2015).

In the past two decades, alternative researches on managing crustacean wastes has been

more focused on the use of enzymes to degrade crustacean waste. Proteolytic enzymes

such as alcalases and trypsin (Synowiecki and Al-Khateeb., 2000), peptidases (Duarte

de Holanda and Netto, 2006), and Triton X-100 (Mizani and Aminlari, 2007) have

been used for hydrolysis on crustacean wastes. Enzymatic hydrolysis is certainly much

more eco-friendly than the use of chemicals. However, commercial enzymes are costly

to obtain and thus making it unsuitable for cost efficient studies (Prameela et al., 2012).

Microbial fermentations were found to be the most flexible, eco-friendly, and

economically viable method (Bashkar et al., 2007). Continuous supply of enzymes is

certain with microorganism fermentation technique; unlike the usage of commercial

enzymes where the enzyme capability has its limit (Synowiecki and Al-Khateeb, 2000).

Instead of enzymatic hydrolysis which only lasts a few hours and may have incomplete

hydrolysis, the continuous production of enzyme in microbial fermentation gives a

more complete hydrolysis which gives a higher yield of recovery in the compound of

interest (Jo et al., 2008). However, microbial fermentation usually takes a longer

completion time as compared to enzymatic hydrolysis.

In the interest of deproteinizing and demineralizing crustacean wastes for astaxanthin,

lactic acid fermentation has shown great success in recovering the pigment (Khanafari

et al., 2007). Lactic acid producing microbes has an advantage in demineralizing

crustacean wastes due to the production of lactic acid during fermentation (Pacheco et

al., 2009). The acid has the ability to dissolve the calcium present in the shells causing

demineralization and deproteinization to occur (Xu et al., 2008). However, this might

seems to be a disadvantage in non-lactic acid producing microbes. Yet, non-lactic acid

producing microbial fermentation has shown similar results in deproteinizing shrimp

and crab wastes (Giyose et al., 2010; Oh et al., 2007). Being that astaxanthin is usually

present in shrimp exoskeleton as carotenoproteins, carotenolipoproteins or

chitinocaroenoids which is a stable form (Sachindra, 2003). Deproteinization is

indispensably required to remove the protein-astaxanthin bond. Possessing other

enzymes or properties for non-lactic acid bacteria add an advantage to the fermentation

system; for instance chitinase for dechitinization. Not only that, enzymes from the

current bacteria might have similar potential with lactic acid fermentation as they

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showed similar deproteinizing results. In terms of cost, lactic acid bacteria are more

costly to be maintained than aerobic bacteria.

Until now, the knowledge on aerobic bacteria fermentation on recovery of astaxanthin

from crustacean wastes is lacking. Aerobic fermentation of crustacean wastes is mainly

focused on the recovery of chitins while the recovery of astaxanthin was less exploited.

The only knowledge of astaxanthin recovery from shrimp shell wastes were by

fermentations with lactic acid producing bacteria. Given that bacteria are ubiquitous in

our environment (Earl et al., 2008), they are cheap and easy to obtain. Therefore, this

provides a route for exploration on the possible recovery of astaxanthin by aerobic

bacteria fermentation. Moreover, the maintenance of microbes is less costly and

environmentally friendly. The addition of optimization to the microbe culturing media

may aid in enhancing its potential in astaxanthin recovery. In optimization studies

conducted by Nisha and Divakaran, (2014), enzyme activity was significantly

increased which eventually increases the activity of crustacean waste degradation.

Meanwhile crustaceans having their carapaces calcified (Finlay et al., 2009) may cause

difficulties to be broken down. Since the current study lack of natural acid production

during fermentation, therefore the addition of cell disruption may aid in decalcification

or demineralization of these shell wastes rendering bond loosening. Traditionally, cell

disruptions were conducted on algae/yeast (Foncesca et al., 2011) and plant cells to

assist inner cell substance extraction by breaking down the cell wall.

Therefore, the aim of the present study is to recover astaxanthin from shrimp shells

through fermentation with aerobic bacteria which produces chitinase and protease.

Both of these enzymes are required due to the presence of carotenoproteins and

interlinks between chitins and proteins (Wang et al., 2007). In addition, shrimp shells

are calcified and the selected bacterium may not produce natural acids to act as

decalcifying agent. Therefore, the selection of a few basic cell disruption techniques

may aid the process of astaxanthin extraction by removing minerals and possible

calcium. Cell disruption techniques were chosen based on its success in extracting

astaxanthin from algae and yeast cells (Xiao et al., 2008; Mendes-Pinto et al., 2001).

Unfortunately, its effectiveness on shrimp shells was yet to be determined as the shells

were not living cells. So far there were not many records on optimizing both enzyme

productions (namely chitinase and protease) in aid of astaxanthin recovery in addition

to aerobic microbial fermentation. Due to that non-lactic acid fermenting bacteria are

chosen for this study, both enzymes are crucial in deproteinization and dechitinization

of the wastes. Although the knowledge on astaxanthin purification from shrimp shells

has been well known, but the purification of pigment resulted from aerobic microbial

fermentations on shrimp shell wastes was yet to be fully explored. With the current

knowledge and technology, purification of astaxanthin from microbial fermentation

possesses certain challenges in obtaining and maximizing the yield of the pigment.

Astaxanthin exists as a complex carotenoid structure in shrimps and is less likely to be

oxidized. By using a green approach, this research focuses on releasing intact

astaxanthin where the ultimate goal is to recover astaxanthin from shrimp shell wastes

using aerobic bacteria. In order to achieve this, isolations were carried out to search for

suitable deproteinizing and dechitinizing bacteria. The bacteria obtained may not be

lactic acid producing and therefore requires external shell disruptions to further remove

calcium and minerals present in the wastes. Selected shell disruption methods were

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non-chemical based which cause less harm to the environment. Deproteinization and

dechitinization are crucial in obtaining astaxanthin hence, optimization of the culture

media and conditions were investigated to increase enzyme activities. An increase in

enzyme activity leads to an increase in the amount of recoverable astaxanthin. This

pigment will be extracted and purified with the common chromatography techniques

(Thin Layer Chromatography and High Performance Liquid Chromatography) to

confirm its presence through this aerobic fermentation system.

In order to achieve the present goal, four objectives were laid out as follows:

1. To isolate and identify bacteria from shrimp shell waste areas that produce

maximum chitinase and protease

2. To evaluate the effectiveness of shell disruptions on astaxanthin recovery

3. To optimize growth media, pH, and temperature for optimal enzyme production

and astaxanthin recovery

4. To quantify and validate astaxanthin recovered from the present microbial

approach.

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BIODATA OF STUDENT

Born in the heart of Malaysia on November 5th, 1988; Cheong Jee Yin obtained her

primary and secondary education from SK Convent Bukit Nanas Kuala Lumpur. Upon

completing her secondary studies, she was offered a 3 years course by Universiti Putra

Malaysia to further her studies in the field of Biology. During her final year of study in

UPM, she was registered under Assoc. Prof. Dr. Muskhazli Mustafa to complete a final

year project entitled “Deproteinization of crustacean shells wastes by Bacillus subtilis

14893”. In 2011, she obtained a Bachelor’s Honours Degree in Biology.

Upon graduation she was offered a Master’s degree by the same undergraduate lecturer

which she accepted the offer. In 2013, she successfully converted her Master degree

studies into Doctoral degree. Within her years of study she has published two journal

articles in her work field. Working on similar background of research, microbes and

astaxanthin has been her passion of research.

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LIST OF PUBLICATIONS

Published Journals

Cheong, J.Y., Nor Azwady, A.A., Rusea, G., Noormasshela, U.A., Nurul Shaziera,

A.G., Azleen, A.A. and Muskhazli M. 2014. The availability of astaxanthin

from shrimp shell wastes through microbial fermentations, Aeromonas

hydrophila and cell disruptions. International Journal of Agriculture and

Biology. 16: 277-284.

Cheong, J.Y., Muskhazli, M., Nor Azwady, A.A., Rusea, G., and Azleen A.A. 2013.

Enhancement of protease production by the optimization of Bacillus subtilis

culture medium. Malaysian Journal of Microbiology. 9: 43-50.

Noormasshela, U. A., Nurul Shaziera, A. G., Cheong, J. Y., Yakubu, S., Azleen, A.

A., Nor Azwady, A. A., and Muskhazli, M. 2015. Toxicity of Bacillus

thuringiensis biopesticide produced in shrimp pond sludge as alternative

culture medium against Bactrocera dorsalis (Hendel). Acta Biologica

Malaysiana. 4: 5-16.

Yakubu, S., Nor Azwady., A.A., Zakaria, M.P., Normasshela, U.A., Nurul Shaziera,

A.G., Cheong, J.Y., Azleen, A.A. and Muskhazli M. 2014. Evaluation of

pH and temperature effects on mycoremediation of phenanthrene by

Trichoderma sp. Acta Biologica Malaysiana. 3: 35-42.

Muskhazli, M., Normasshela, U., Cheong, J.Y., Nor Azwady, A.A., and Rusea, G.

2012. Purification of Chitinase 33kDa and expression pattern of chit33 in

Trichoderma longibrachiatum T28. Acta Biologica Malaysiana. 1: 18.

List of Proceedings

Cheong, J.Y., Muskhazli, M., Nor Azwady, A. A., Siti Aqlima, A., and Azleen, A. A.

2014. Extraction and purification of astaxanthin by Aeromonas hydrophila.

In Proceeding of the Malaysia International Biological Symposium, 28-29th

October 2014, Selangor, Malaysia.

Cheong, J.Y., Nor Azwady, A.A, Rusea, G., Noormasshela, U.A., Nurul Shaziera,

A.G., Safiya, Y., Azleen, A.A and Muskhazli, M. 2013. Recovery of

Astaxanthin from Shrimp Shells by Aeromonas hydrophilia and Mechanical

Cell Disruption. In Proceeding of the 5th Fundamental Science Congress,

20-21st August 2013, Selangor, Malaysia. pp 98-101.

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Noormasshela, U.A., Nurul Shaziera A.G., Cheong J.Y., Yakubu, S., Azleen A.A.,

Nor Azwady A.A., and Muskhazli, M. 2013. Toxicity of Bacillus

thuringiensis biopesticide produced in shrimp pond sludge as alternative

culture medium against Bactrocera dorsalis (Hendel). In Proceeding of the

5th Fundamental Science Congress, 20-21st August 2013, Selangor,

Malaysia. pp 93-97

Nurul Shaziera, A.G., Nor Azwady, A.A., Noormasshela, U.A., Cheong, J.Y.,

Yakubu, S., Azleen, A.A., and Muskhazli, M. 2013. Assessment of

Ganoderma boninense PER 71 as saccharification agent based on

lignocellulolytic enzymes activities from pretreated paddy straw

hydrolysate. In Proceeding of the 5th Fundamental Science Congress, 20-

21st August 2013, Selangor, Malaysia. pp 102-106.

Yakubu, S., Nor Azwady, A.A., Zakaria, M.P., Noormashela, U.A., Nor Shaziera,

A.G., Cheong, J.Y., Azleen, A.A., and Muskhazli, M. 2013. Application of

fungi isolated from soil for PAH (phenanthrene) degradation. In Proceeding

of the 5th Fundamental Science Congress, 20-21st August 2013, Selangor,

Malaysia. pp 223-226.

Cheong, J.Y., Muskhazli, M., Nor Azwady, A. A., and Azleen A. A. 2012.

Extracellular neutral chitinase and protease production from a newly

isolated bacteria. In Proceeding of the Malaysia International Biological

Symposium, 11-12th July 2012, Selangor, Malaysia. pp 101

Cheong, J.Y., Muskhazli, M. And Nor Azwady, A.A. 2011. Deproteinization of

crustacean waste shell by Bacillus subtilis ATCC 14893. In Proceedings of

the 4th Regional Fundamental Science Congress, 5-6th July 2011, Selangor,

Malaysia. pp 177-178.

universiti putra malaysia isolation, optimization and ...psasir.upm.edu.my/id/eprint/66866/1/fs 2016...

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